| 1. |
- Junker, Johan, 1980-
(författare)
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Human Dermal Fibroblasts in Tissue Engineering
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The loss or failure of tissues and/or organs is one of the most frequent problems in modern healthcare. The field of tissue engineering applies the principles of biology and engineering in order to develop functional substitutes for damaged tissues. Tissue engineering contains elements of medicine, material science and engineering with major components in focus being cells, biomaterials and soluble factors. All three components may be required for the development of clinical treatments.The usage of autologous tissue specific cells for clinical treatment is often not feasible due to poor growth kinetics or unstable phenotypes of the cells. Furthermore, lack of availability of healthy tissue that can be biopsied is a major problem in many applications. One approach to overcome this problem is to use adult stem cells which have the capacity to give rise to several different cell types. Although promising, adult stem cells have major impediments for use in several tissue engineering applications. The difficulties associated with harvest, culture and storage render problems in the development of clinically relevant procedures.During the last years, the inherent plasticity of differentiated somatic cells has been demonstrated. One of the easiest human cell types to obtain, expand and store is the dermal fibroblast. Recent reports indicate that dermal fibroblasts can be induced to differentiate towards several distinct mesenchymal lineages in vitro.The main aim of this thesis was to investigate the inherent stem cell plasticity of human dermal fibroblasts and explore their possible usefulness in tissue engineering applications. The papers included in this thesis employ routine and immunohistochemical staining, enzyme activity assay, analysis of low density lipoprotein incorporation, capillary-like network formation assay and full expression micro array analysis.Fibroblasts were shown to differentiate towards adipocyte, chondrocyte, endothelial and osteoblast-like cell types in vitro. The differentiation from fibroblasts to myofibroblasts in burn scar tissue upon stimulation by mechanical tension was also demonstrated. Adipogenic, chondrogenic and osteogenic induced fibroblasts display the upregulation of several genes associated with adipocytes, chondrocytes and osteoblasts.
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| 2. |
- Lönnqvist, Susanna, 1986-
(författare)
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Applications of human skin in vitro
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic wounds are a substantial problem in today’s health care and place significant strains on the patient. Successful modelling of the wound healing process is pivotal for the advancement of wound treatment research. Wound healing is a dynamic and multifactorial process involving all constituents of the skin. The progression from haemostasis and inflammation to proliferation of epidermal keratinocytes and dermal fibroblasts, and final scar maturation can be halted and result in a chronic wound that fails to re-epithelialise. The wound healing process constitutes an example of dynamic reciprocity in tissue where cellular changes take place on cues from the extracellular matrix and vice versa when tissue homeostasis is disturbed. The extracellular matrix provides a structural context for the resident cells and the epidermal keratinocytes, and a functioning interplay between the two tissue compartments is crucial for successful wound healing to take place. Work included in this thesis has applied viable human full thickness skin in vitro to investigate the re-epithelialisation process and barrier function of intact skin.The use of full thickness skin in vitro can take into account the contextual aspect of the process where the epidermal keratinocytes are activated and obtain a migratory phenotype, and are continuously dependent on the cues from the extracellular matrix and support of the dermis. When utilising skin for studies on re-epithelialisation, circular standardised full thickness wounds were created and cultured for up to four weeks in tissue culture. In paper I, the organisation of a thick neoepidermis was investigated in the in vitro wound healing model when resident cells were provided with a porous suspended three dimensional gelatin scaffold. In paper II we investigated the use of a fluorescent staining conventionally used for proliferation studies to facilitate the tracing of transplanted epidermal cells in in vitro wounds, in order to improve and expand the use of the model. In paper III the model was utilised to investigate the treatment approach of acidification of wounds to evaluate the suitability of such intervention in regards to keratinocyte function and re-epithelialisation. Studies on re-epithelialisation with the aid of the in vitro wound healing model provided insight in neoepidermal structure with porous gelatin scaffolding in the wound, a novel methodological approach to tracing cells and response to constrained wound healing environment. In paper IV, intact human skin was evaluated for modelling the cytotoxic response after exposure to a known irritant compound. To study barrier function, intact skin was exposed to irritants by restricting exposure topically, and full thickness skin in vitro was found suitable for modelling cytotoxicity responses. Employing human full thickness skin in vitro makes use of the actual target tissue of interest with epidermal and dermal cells, and full barrier function.
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| 3. |
- Nyman, Erika, 1976-
(författare)
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Guided Regeneration of the Human Skin : in vitro and in vivo studies
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Every day and in all parts of the world, humans experience different grades of wounding and tissue loss of the skin, thus initiating one of the most complex biological processes. Acute and chronic wounds, as well as the additional problem of skin scarring, involve not only great suffering for the patient but also extensive health care costs for the society. Although the wound-healing process is a wellstudied field much knowledge must be gained to unlock the door to regenerative pathways in humans.Epidermis heals by complete regeneration, but dermal and full thickness injuries heal with fibrosis and scar formation. In Papers I and II, we studied whether dermal scarring could be turned into regeneration by using two different types of threedimensional dermal scaffolds. In Paper I, we studied a solid scaffold made of poly(urethane urea), initially in vitro then followed by in vivo studies. In Paper II, we intradermally injected a liquid three-dimensional scaffold consisting of porous gelatin spheres in human healthy volunteers. Both materials showed ingrowth of functional fibroblasts and blood vessels and appeared to stimulate regeneration while slowly degrading. This finding could be of significant clinical importance, for example in burn wound care or after cancer surgery.In Papers III and IV, we wanted to study the effects of amniotic fluid and hyaluronic acid on adult wound healing, because early fetal wounds re-epithelialize rapidly and naturally heal dermis by regeneration without the need of a dermal scaffold. Amniotic fluid, naturally rich in hyaluronic acid, induced an accelerated reepithelialization of adult human wounds in vitro, and hyaluronic acid seemed to be important for this effect. Stimulation with exogenous hyaluronic acid in vivo induced accelerated re-epithelialization and an altered protein expression in healthy human volunteers. The inflammatory phase of wound healing, as measured by tissue viability imaging, was not affected by hyaluronic acid. Elucidating the effects of amniotic fluid and hyaluronic acid on the wound-healing process may allow improved treatment of wounds with impaired healing.Studies on finding new dermal scaffolds and studies on the positive effect of amniotic fluid or hyaluronic acid on the wound-healing process are two different ways of gaining insight that may lead to regeneration and improved wound healing for the patient.
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| 4. |
- Pettersson, Sofia, 1977-
(författare)
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Biodegradable gelatin microcarriers in tissue engineering : In vitro studies on cartilage and bone
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tissue engineering is a multidisciplinary field that combines cells, biomaterial scaffolds and environmental factors to achieve functional tissue repair. This thesis focuses on the use of macroporous gelatin microcarriers as scaffolds in tissue engineering applications, with a special focus on cartilage and bone formation by human adult cells in vitro.In our first study, human articular chondrocytes were seeded on macroporous gelatin microcarriers. The microcarriers were subsequently encapsulated in coagulated blood-derived biological glues and cultured under free-swelling conditions for up to 17 weeks. Even in the absence of recombinant chondrogenic growth factors, the chondrocytes remained viable and metabolically active for the duration of the culture period, as indicated by an increased amount of cell nuclei and extracellular matrix (ECM). The ECM showed several cartilage characteristics, but lacked the cartilage specific collagen type II. Furthermore, ECM formation was seen primarily in a capsule surrounding the tissue-engineered constructs, leading to the conclusion that the used in vitro models were unable to support true cartilage formation.The capacity of human dermal fibroblasts to produce cartilage- and bone-like tissue in the previously mentioned model was also investigated. Under the influence of chondrogenic induction factors, including TGF-β1 and insulin, the fibroblasts produced cartilage specific molecules, as confirmed by indirect immunohistochemistry, however not collagen type II. Under osteogenic induction, by dexamethasone, ascorbate-2-phosphate and β–glycerophosphate, the fibroblasts formed a calcified matrix with bone specific markers, and an alkaline phosphatase assay corroborated a shift towards an osteoblast like phenotype. The osteogenic induction was enhanced by flow-induced shear stress in a spinner flask system.In addition, four different types of gelatin microcarriers, differing by their internal pore diameter and their degree of gelatin cross-linking, were evaluated for their ability to support chondrocyte expansion. Chondrocyte densities on the microcarriers were monitored every other day over a twoweek period, and chondrocyte growth was analyzed by piecewise linear regression and analysis of variance (ANOVA). No differences were seen between the different microcarriers during the first week. However, during the second week of culture both microcarrier pore diameter and gelatin crosslinking had significant impacts on chondrocyte density.Lastly, a dynamic centrifugation regime (f=12.5 mHz for 16 minutes every other day) was administered to chondrocyte-seeded microcarriers, with or without encapsulation in platelet rich plasma (PRP), to study the possible effect of dynamic stimuli on cartilage formation. Presence of PRP enhanced the structural stability of the tissue-engineered constructs, but we were not able to confirm any dose-response pattern between ECM formation and the applied forces. After 12 weeks, distinct gelatin degradation had occurred independent of both dynamic stimuli and presence of PRP.In summary, this thesis supports a plausible use for gelatin microcarriers in tissue engineering of cartilage and bone. Microcarrier characteristics, specifically gelatin cross-linking and pore diameter, have been shown to affect chondrocyte expansion. In addition, the use of human dermal fibroblasts as an alternative cell source for cartilage and bone formation in vitro was addressed.
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| 5. |
- Rakar, Jonathan
(författare)
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Fibroblast Differentiation and Models of Human Skin
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis combines three publications and one manuscript, covering two principal topics: functional differentiation of human fibroblasts and laboratory models of human skin. The two topics favourably unite in the realm of tissue engineering. This thesis is therefore split into three main parts: 1. a discussion of phenotypic plasticity as it pertains to fibroblasts and the stem cell continuum; 2. a short review of engineered tissue, with particular focus on soluble factors and materials; and, 3. a motivated review of the biology, diversity and culture of skin, including skin construction.The intended goal of our research endeavor was to achieve the formulation of a bioactive therapy for skin regeneration. The main hypothesis was that fibroblast-to-keratinocyte differentiation would facilitate wound healing, and that the protocol for such a method could be adapted to clinical translation. The foundation for the hypothesis lay in the differentiation capabilities of primary dermal fibroblasts (Paper I). However, the goal has not yet been achieved. Instead, intermediate work on the construction of skin for the purpose of creating a model test-bed has resulted in two other publications. The use of excised human skin, a formidable reference sample for tissue engineered skin, has been used to investigate a gelatinbased material in re-epithelialization (Paper II). A first attempt at standardizing a constructed skin model also resulted in a publication: an evaluation of melanocyte influences on keratinocyte-mediated contraction (Paper III).The introduction of melanocytes into a skin model raised questions about other appendages of the integumentary system. Our previous experience with preadipocyte isolation and identification, and our attempts at constructing three-dimensional adipose tissue, motivated further investigations into fibroblast-to-adipocyte differentiation. We investigated the possibility of activating thermogenesis in fibroblasts, a property otherwise reserved for cells of the adipogenic and myogenic lineages. Our attempts were successful, and are presently in manuscript form (Paper IV). Some further experiments and optimizations are necessary before establishing a reproducible protocol for thermogenic induction.The knowledge obtained through these scientific inquiries have moved us closer to achieving our goals, but methodological advances are still necessary. In the meantime, we have new test-beds for investigating different interactions in skin, and that enables many new questions to be asked and answered.
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| 6. |
- Sommar, Pehr
(författare)
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Differentiation of Human Dermal Fibroblasts and Applications in Tissue Engineering
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors.One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin.The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications.Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV).In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.
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| 7. |
- Svernlöv, Birgitta
(författare)
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Treatment of Epicondylalgia and Nerve Entrapments around the Elbow
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Disorders causing pain in the elbow region is a common problem and is one of the most frequent forms of work-related health problems. These conditions are thus of major importance from the public health point of view, as well as from that of the suffering individual. Both sensory and motor function may be impaired, particularly in cases where a nerve is involved, resulting in severely impaired hand function.“Tennis elbow” (lateral epicondylalgia) has been found to be the second most frequently diagnosed musculoskeletal disorder of the upper extremity in the primary health-care setting. “Golfer’s elbow” (medial epicondylalgia) is not that commonly encountered. It has been stated that tennis or golfer’s elbow syndromes are self-limiting. Even so, clinical experience has shown that there are a few cases where symptoms have a painful and long-lasting course, resistant to many forms of therapy. The outcomes of frequently adopted management regimes for treatment of epicondylalgia or nerve entrapments around the elbow were examined in the following five studies:I: A randomised, prospective study of 38 patients with tennis elbow (lateral epicondylalgia). Groups were assigned to eccentric exercises or stretching. Eccentric exercise gave somewhat better results.In a second part of the study, a 4-year follow-up of 127 patients who used eccentric exercises for tennis elbow was performed. Patients showed decreased pain and increased grip-strength after 3 months treatment. At the time of publication this study was the first to examine eccentric exercises for this condition.II: A retrospective analysis of long-term results from 51 patients treated with surgical release of the common extensor origin because of “chronic tennis elbow”. Eighty-seven per cent of the patients rated themselves as completely recovered or improved.III: A randomised, prospective study of 70 patients with ulnar neuropathy in the forearm (cubital tunnel syndrome). Groups assigned the commonly recommended elbow brace at night or nerve gliding exercises were compared with a control group (information only). The majority of patients improved considerably, both subjectively and objectively, after a 3 months period, regardless of group. The study thus showed the effectiveness of information and expectance, and that orthosis or nerve gliding exercises added nothing further to the result.IV: A retrospective study of 205 patients treated with surgery for suspected nerve entrapment in the forearm. Followup, almost 4 years later, demonstrated a subjective improvement in two of three patients, but only 3% experienced complete relief of all symptoms.V: A prospective long-term study on 20 patients with golfer’s elbow (medial epicondylalgia) treated with eccentric exercises over 3 months. The results showed decreased pain and increased gripstrength. This is the first study published on the management of this disorder with eccentric exercises.
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